Molecular recognition, such as between receptor ligand, antigen-antibody, DNA-protein, sugar-lectin, RNA-ribosome, etc. is an important principle underlying many biological systems and is being applied to many artificially created biological systems for use in medical applications, such as in artificial (micro- or nano-) particulate systems including polymeric beads, vesicular lipids, microemulsions, and the like.
One important example of a molecular recognition based application is the use of targeted delivery of diagnostic or therapeutic compounds, such as antiviral, chemotherapeutic or imaging agents, to specific sites, which allows to overcome the limitations associated with nonspecific delivery (such as in vivo clearance time, potential toxicity, problems associated with membrane transport of an agent and the like) and thus greatly increases their effectiveness. Various recognition-based strategies have been used to improve the delivery of compounds into the intracellular environment (i.e. to specific cell compartments) of a target cell to exert its biological activity, in particular delivery through specific transporters involving the use of biological or artificial carriers, such as viral vectors, cationic polymers, such as polylysine, polyarginine and the like (see, e.g. WO 79/00515, WO 98/52614), lipid carriers, and various other conjugate systems.
One widely used approach involves the use of lipid vesicles as artificial carriers, e.g. liposomes and micelles, which have been extensively developed and analyzed as drug delivery vehicles due to their ability to reduce systemic exposure of a bioactive agent, thereby overcoming problems associated with degradation, solubility, etc. and providing an increase in blood circulation times. Actively targeted delivery of a bioactive agent involves derivatizing the lipids of the lipid vesicle (either prior or after vesicle formation) with a targeting ligand that serves to direct (or target) the vesicle to specific cell types such as cancer cells or cells specific to particular tissues and organs, such as hepatocytes, after in vivo administration (see, for example, U.S. Pat. No. 6,316,024 and U.S. Pat. No. 6,214,388; Allen et al., Biochim. Biophys. Acta, 1237:99-108 (1995); Blume et al., Biochim. Biophys. Acta, 1149:180-184 (1993)). This may be accomplished by utilizing receptors that are overexpressed in specific cell types, which include for example folic acid receptor (FR) (overexpressed in a variety of neoplastic tissues, including breast, ovarian, cervical, colorectal, renal, and nasoparyngeal tumors), transferrin receptor (TfR) (overexpressed on metastatic and drug resistant cells of most carcinomas, sarcomas and some lymphomas and leukaemias), epidermal growth factor receptor (EGFR) (overexpressed in anaplastic thyroid cancer and breast, lung and colorectal tumors), vascular endothelial growth factor receptor 1 and 2 (VEGFR-1/2) (highly expressed on endothelial cells in tumor neovasculature), metastin receptor (overexpressed in papillary thyroid cancer), ErbB family receptor tyrosine kinases (overexpressed in a significant subset of breast cancers), human epidermal growth factor receptor-2 (Her2/neu) (overexpressed in breast cancers), tyrosine kinase-18-receptor (c-Kit) (overexpressed in sarcomatoid renal carcinomas), HGF receptor c-Met (overexpressed in esophageal adenocarcinoma), CXCR4 and CCR7 (overexpressed in breast cancer), endothelin-A receptor (overexpressed in prostate cancer), peroxisome proliferator activated receptor delta (PPAR-delta) (overexpressed in most colorectal cancer tumors), PDGFR A (overexpressed in ovarian carcinomas), BAG-1 (overexpressed in various lung cancers), soluble type II TGF beta receptor (overexpressed in pancreatic cancer), asialoglycoprotein receptor (overexpressed on hepatocytes), αvβ3 integrin receptor (overexpressed in growing tumor vascularture), legumain (a clan CD cysteine protease enriched in solid tumor tissue and overexpressed on TAMs, tumor associated macrophages), etc.
Any agent which selectively binds to such a specific receptor cell or tissue to be treated or assayed may be attached to a lipid vesicle and act as a targeting or receptor ligand. Typically, such targeting ligands have been attached to a lipid or lipid vesicle surface through a long chain (e.g. polymeric) linker. For example folic acid based conjugates have been used to provide a targeted delivery approach of a therapeutic compound useful for the treatment and/or diagnosis of a disease, allowing a reduction in the required dose of therapeutic compounds (see e.g. WO 02/094185, U.S. Pat. No. 6,335,434, WO 99/66063, U.S. Pat. No. 5,416,016). Likewise, the use of galactose- and galactosamine-based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease such as HBV and HCV infection or hepatocellular carcinoma while allowing a reduction in the required dose of therapeutic compounds required for treatment (see e.g. U.S. Pat. No. 6,030,954, . . . ).
Another important example of a molecular recognition based application is the use of antigen display systems which involve presentation of both “self” and “foreign” proteins (antigens) to the immune system to generate T cell activation, modulation or tolerance. The receptor ligand interactions in antigen-presenting systems that contribute to the desired immune response or absence thereof are complex and difficult to assess, being influenced by various parameters such as ligand densities, presence of coreceptors, receptor ligand affinities and surface conditions. Thus a widely used approach involved using naturally occurring human cells (or parts thereof) whose primary function is antigen processing and presentation. But, while live cell based systems may be optimal for mimicking cell-cell interaction to achieve the desired induction of tolerance or immune response, they are dependent on a regulated expression of the surface molecules including possibly expression of additional “costimulatory” and/or adhesion molecules on its surface membrane at a sufficient therapeutic level. Currently known artificial systems range from genetically engineered subcellular antigen presenting vesicles, which carry the molecules required for antigen presentation and T-lymphocyte activation or inhibition on their surface (WO 03/039594) to systems on the basis of cell-sized, biodegradable microspheres based, antigen presenting system (WO 07/087341).
Clearly, there are still drawbacks to the above, molecular recognition based technologies and there remains a need in the art for a versatile and efficient artificial carrier system for use in molecular recognition based applications such as targeted delivery or antigen presentation, including simple and economic methods of their preparation.
The present application provides conjugates comprising ether-lipids having one or more covalently attached bioactive ligands as well as various carrier systems comprising these conjugates (and optionally further comprising one or more bioactive agents), which allow to overcome the limitations described above.